1. Field of the Invention
The present invention is directed to novel electrochemiluminescence (ECL) reactions and assays based on such reactions for the determination of soluble metal ions and diaminoaromatic ligands. Specifically, the present invention is directed to a method for detecting diaminoaromatic ligands and soluble metal ions in, and for environmental analysis of, industrial wastewater streams, soils, groundwater supplies and the like. Furthermore, the present invention is directed to a method for determining atomic or intermolecular distances, for studying electronic states of molecules and characterizing coordinate bonding of complexes that exhibit ECL upon binding.
2. Description of the Prior Art
Trinitrotoluene (TNT), other nitroaromatics and their byproducts or degradation products, especially the aminoaromatics, are significant military and industrial pollutants in terms of their toxicity, distribution, and relative abundance and are consequently of environmental concern. Likewise, many of the heavy and transition metals are highly toxic industrial pollutants. Assays and environmental sensors for toxic organics, such as TNT and metals, are continuously being developed and improved to yield better sensitivity and reliability for use in complex matrices as well as design simplicity and assay execution under field conditions.
Chemiluminescent (CL) assays have demonstrated extreme sensitivity, which in some cases rivals that of radioisotopic methods. Electrochemiluminescence (ECL) is a form of chemiluminescence which involves inducing luminescence via application of a voltage potential. ECL has been applied to such diverse problems as sensitive detection of biotoxins, nucleic acids, and bacterial pathogens. ECL has a distinct advantage over other forms of CL in that the luminescence can be controlled by the applied voltage at the working electrode.
ECL molecules can be entirely organic, organic-metal ion coordination complexes, or other types of molecules. An article entitled "Occurrence, Mechanism and Analytical Applications of Electrogenerated Chemiluminescence", authored by A. W. Knight and G. M Greenway, Analyst, (1994) Vol. 119, pp.879-890, the disclosure of which is expressly incorporated herein by reference, discusses various known ECL compounds. Four main chemical systems which are known are referred to. The first system is ECL of polyaromatic hydrocarbons in both non-aqueous and aqueous media. The second is methods based on luminol reaction in alkaline solution. The third concerns ECL reactions using ruthenium (II) tris (2,2'-bipyridine), in particular its use as an ECL label for other non-electrochemiluminescent compounds or for the quantification of persulfates and oxalates. Finally, the fourth known ECL system discussed involves analytical aspects of cathodic luminescence at an oxide covered aluminum electrode.
Ruthenium trisbipyridine (Ru(bpy).sub.3) is, perhaps, the best known of the organic-metal ion coordination complex ECL molecules, although other transition metal complexes involving Cr, Cu, Eu, Ir, Mg, Os, and Tb have been observed to exhibit ECL under appropriate conditions.
Ru(bpy).sub.3 based ECL resembles fluorescence and phosphorescence in that an excited electronic state is induced in the ECL molecule which subsequently relaxes to a ground state to yield light. In the model Ru(bpy).sub.3.sup.+2 ECL system, upon which the well known ORIGEN Technology is based (IGEN International Inc., Gaithersburg, Md.), Ru(bpy).sub.3.sup.+2 is oxidized to the +3 state at the surface of an anode. Subsequently, Ru(bpy).sub.3.sup.+3 is placed in an excited state (Ru(bpy).sub.3.sup.+2.cndot.) by a high energy free radical electron transfer from an electron carrier such as nascent TPA.sup.+.cndot. radical, which is generated simultaneously at the anode. Relaxation of the high energy Ru(bpy).sub.3.sup.+2.cndot. to the ground state yields luminescence at approximately 602 nm.
ORIGEN Technology is well known in the art and has been used to perform ECL immunoassays. Such immunoassays are generally performed by mixing sample fluid with two antibodies, one labeled or tagged with magnetic beads and the other labeled with ruthenium (II) tris (bipyridyl). After a short incubation, the amount of analyte is quantified by the emission of light from the ruthenium label or tags.
ECL is essentially a surface redox reaction which allows ECL molecules, such as Ru(bpy).sub.3.sup.+2, primarily near the surface of the anode to undergo the ECL redox reaction sequence. This is an advantage in immunomagnetic bead-ECL assays as the overlying fluid phase contributes only negligible ECL compared to the magnetically captured antigen-antibody-ECL tag complexes on the anode surface, thus obviating "wash" steps. However, the surface redox phenomenon is a disadvantage for "solution phase" ECL measurements as sensitivity may be somewhat compromised. Thus, the use of immobilized ECL complexes, while not completely necessary, is probably desirable. U.S. Pat. No. 5,324,457, the disclosure of which is expressly incorporated herein by reference, describes devices and methods for generating electrogenerated chemiluminescence primarily with ruthenium complexes. U.S. Pat. Nos. 5,221,605 and 5,238,808, also expressly incorporated herein by reference, are directed to ECL reactions involving ruthenium or osmium complexes and their use as labels in binding methods for determining the presence of substances of interest.
Prior to the present invention ECL reactions based on specific metal ions being captured by organic ligands to form ECL compounds, according to the present invention, had not been contemplated. Detection and identification of specific metal ions is traditionally carried out by flame or furnace atomic spectroscopy which can be costly and slow. Detection of organics is traditionally carried out by gas chromatography (GC), high performance liquid chromatography (HPLC) and mass spectroscopy (MS) devices which is usually costly and the instrumentation required to carry out such determinations can be large, at least in comparison to that required for ECL reactions.